Prognostic value of tumor‐infiltrating lymphocytes and PD‐L1 expression in esophageal squamous cell carcinoma

Abstract Background Tumor cells (TC) participate in tumor progression by altering the immune responses in the tumor microenvironment. However, the clinical relevance and prognostic effect of PD‐L1 expression and tumor‐infiltrating lymphocytes (TILs) in esophageal squamous cell carcinoma (ESCC) are unknown. The purpose of this study was to investigate the interactions and clinical significance of PD‐L1 expression and TILs in ESCC. Methods Tissue specimens were collected from 126 patients with ESCC who underwent curative esophagectomy. Immunohistochemical analysis and multiplex immunofluorescence for CD4, CD8, CD25, FOXP3, and PD‐L1 in the tumor were used to identify multiple tumor‐infiltrating immune cells (TIIC), Tregs, and TC. Results PD‐L1 was expressed in tumor cells (PD‐L1 TC). PD‐L1 TIIC and PD‐L1 TC affected the biological behavior of TC. The positive expression rate of PD‐L1 TC and CD8+ TILs was 27.8% (35/126) and 31.7% (40/126), respectively. Kaplan–Meier analysis showed that overall survival (OS) was significantly associated with decreased CD8+ TILs and PD‐L1 TC‐positive expression, which promote ESCC progression and metastasis. Conclusion Tumor depth, CD8, and PD‐L1 TC were independent prognostic factors in ESCC, and a predictive nomogram with these three risk factors improved the accuracy of predicting OS in patients with ESCC after surgical resection. The conjoint analysis of multiple immune‐related factors is beneficial for stratifying patient survival risk.

Esophageal cancer (EC) is among the most common malignant tumors of the gastrointestinal tract, ranking seventh in terms of incidence and sixth in mortality. 1Among EC cases, more than 90% are esophageal squamous cell carcinoma (ESCC), with a dismal survival outcome. 2 Despite multidisciplinary therapeutic approaches including immune checkpoint inhibitors, the prognosis for patients with ESCC remains poor. 3he complex tumor microenvironment (TME) involves several tumor-immune interactions, wherein continuous interactions between tumor cells (TC) and immune cells are a predominant feature that affects the immune response and contribute to tumor survival and aggressiveness, even altering patient prognosis. 4,5ccumulating evidence suggests that PD-L1 expressed on TC or immune cells affects the interplay between TC and tumor-infiltrating lymphocytes (TILs) in the TME, attenuating the host anti-tumor immune response. 6,7umoral PD-L1 expression status is closely correlated with the progression of the tumor and provides prognostic information. 8,9PD-L1 expression is closely related to survival in ESCC but its clinical significance remains controversial. 10,11ILs are the most indispensable immune cell component in the anti-tumor immune response in the TME. 12,13ccumulation of CD8 + TILs in tumor tissue correlates with favorable clinical outcomes. 14,150][21] Therefore, it is necessary to comprehensively understand the expression of PD-L1 and TILs in the tumor immune microenvironment of ESCC and their interdependence to explore more reliable prognostic immune factors to predict the prognosis of patients with ESCC.
In the present study, multiple TILs (CD4 + TILs, CD8 + TILs, CD25 + TILs, FOXP3 + TILs, CD4 + CD25 + FOXP3 + Tregs) and PD-L1 expression were assessed in tissues specimens of patients with ESCC who underwent curative esophagectomy without any preoperative therapy to assess their clinicopathological characteristics and prognosis.Clinical prediction models were generated by integrating overall survival (OS)-associated risk factors for ESCC, and the accuracy of the results was tested using calibration curve and time-dependent receiver operating characteristic (time-dependent ROC) curves.

| Patients and sample preparation
Specifically, 126 patients with pathologically confirmed ESCC who underwent radical esophagectomy at Chiba University Hospital were enrolled in our study (January 2001-April 2017), and 24 patients received adjuvant chemotherapy after surgery.The patients who underwent preoperative treatment were excluded.Tumor specimens were fixed in 10% formaldehyde solution, embedded in paraffin blocks, and cut into 4 μmthick serial sections to evaluate immune cell infiltration and PD-L1 expression.The staging classification was assessed using the TNM system (UICC 8th edition). 22The median follow-up time after surgery was 39.4 months (range 2-153 months).OS was defined as the period from the date of surgery to death.This study was approved by the Clinical Research Ethics Committee of Chiba University and informed consent was obtained from all patients to use their surgical specimens for research purposes.

| Immunohistochemistry
Sections were deparaffinized and incubated with Antigen Retrieval Solution (HistoVT One, pH 7.0, Nacalai, Japan) at 95°C for 40 min.Endogenous peroxidase activity was blocked using Peroxidase-Blocking Solution (Dako S2023, Japan) for 30 min.Serial sections were incubated with antihuman CD8 mouse monoclonal antibody (clone: C8/144B; 1/100; Dako), anti-human FOXP3 mouse monoclonal antibody (clone: 236A/E7; cat.no.ab20034; 1/100; Abcam), and anti-human PD-L1 rabbit monoclonal antibody (clone: 28-8; cat.no.ab205921; 1/500; Abcam) overnight at 4°C.Sections were washed with Tris-buffered saline with tween (Dako S3006), and incubated with the corresponding secondary antibody at 37°C for 60 min.After washing, the sections were incubated with 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution (DAB; Dako K3468) at room temperature for 90 s, 25 min, and 90 s.Subsequently, the sections were counterstained with hematoxylin for 1 min, dehydrated using ethanol, mounted, and examined under low-power fields (original magnification ×40) using a Motic microscope (BA210E Upright) to confirm the tumor area and integral dye state.CD8 and FOXP3 were localized on the membranes and in the nuclei of TILs as yellowish-brown and tan signals, respectively.PD-L1 was predominantly present and localized to the membrane or cytoplasm of tumor and immune cells, which were observed as brown signals of differing intensities.

| Evaluation of immunohistochemistry
Five fields with the most adequately stained cells in the tumor center were captured and manually counted under a high-power field (HPF) (magnification ×400) (Figure 1A) to assess CD8 + and FOXP3 + TILs.Each field included at least 200 immune or TC.The calculation was as follows: TILs (%) = positive immune cells/(immune cells + tumor cells) × 100. 23The final maximum value was the corresponding TILs percent.The optimal cutoff values for CD8 + and FOXP3 + TILs were 21% and 8%, respectively.
Tissue sections were independently examined more than twice by two pathologists (H.J. and T.T.) who were blinded to the patient's clinical information.Necrotic areas were excluded from the observation field.In case of a discrepancy, the sections were reviewed by the pathologists together and the decision was made by consensus.

| Evaluation of immunofluorescence
Tumor-infiltrating T lymphocytes in each tissue specimen were observed at low-power fields (original magnification ×100).Three or more of the most abundantly distributed areas in the tumor center were selected, and high-power field (HPF) (original magnification ×400) images were obtained for exact quantification using Image-Pro Plus software.The total number of cells was analyzed for CD4, CD8, CD25, and FOXP3 immunofluorescence staining intensity, and the maximum was recorded.A total of 89 cells/HPF, 111 cells/HPF, and 60 cells/HPF were defined as the optimal cut-offs for CD4 + TILs, CD25 + TILs, and FOXP3 + TILs, respectively, and classified into high and low groups.
The following T cell phenotypes were quantified separately using the identical set of merged images: 1H).CD4 (green signal) and CD25 (red signal) staining were detected on the T-cell membrane, and FOXP3 (blue signal) was observed in the cell nucleus with a typical dotted staining pattern.T cells with the CD4 + CD25 + FOXP3 + phenotype were considered Tregs. 17he optimal cutoff values corresponding to the four T cell phenotypes were 80 cells/HPF, 2 cells/HPF, 1 cell/HPF, and 2 cells/HPF, respectively.

| Statistical analysis
All statistical analyses were performed using GraphPad Prism 10.2.0, SPSS 29.0, and R software (http:// www.r-proje ct.org/ ).Clinicopathological characteristics were analyzed using Chi-Square and Fisher's exact tests.The optimal cutoff values of TIL markers and PD-L1 TC expression were determined by ROC curve analysis.The correlation matrix for the markers was based on Spearman's rank correlation analysis.A two-sided t-test for unpaired samples was used to examine statistically significant differences between PD-L1 and T-cell phenotypes.The Kaplan-Meier curve was used to assess survival.Cox proportional hazards regression was used to identify the prognostic impact of the clinical variables and multiple potential biomarkers.All p-values were two-sided, and a p-value <0.05 indicated a statistically significant result.Nomogram was constructed using the "rms" package in R (version 4.2.2).Calibration analyses were performed to examine the consistency between the risk predicted by the clinical prediction model and the actual risk of occurrence.Time-dependent ROC analysis was performed to compare the ability of specific prognostic indicators to predict OS.

| Correlation between tumor depth, CD8 + TILs, PD-L1 expression, and prognosis in ESCC
Among the clinicopathological characteristics, the median survival of patients ESCC with T1 + 2 and T3 + 4 was 44.6 and 27.0 months, respectively (p = 0.0003, Figure 1B).The OS rate was significantly higher in patients with positive CD8 expression (5-year OS: positive vs. negative: 80.9% vs. 54.4%,p = 0.0028, Figure 1C).Compared with PD-L1 TC-negative patients, the 5-year postoperative survival rate of patients with positive expression was significantly lower (p = 0.0402, Figure 1D).We found no statistically significant difference between PD-L1 TIIC-positive and The bold values in the table indicate that the p-value is statistically significant.

| Predictive nomogram for overall survival of patients with ESCC
Based on the results of the multivariate analysis, tumor depth, CD8, and PD-L1 TC levels were included in the nomogram prediction model to predict the 1-, 3-, and 5-year survival probabilities (Figure 2A).The 3-year calibration curves demonstrated a high consensus between the predicted and actual survival rates, with a Cindex of 0.756 (0.724-0.788), indicating that our predictive model was highly discriminative and accurate (Figure 2B).Time-dependent ROC was used for validation, and the areas under the ROC curve (AUC) for tumor depth, CD8, and PD-L1 TC levels were 0.69, 0.66, and 0.57, respectively (Figure 2C).

CD4 + CD25
The upregulation of PD-L1 affects the development, progression, and prognosis of malignant tumors, 30,31 however, this finding remains controversial in ESCC.Guo et al. 32 suggest that PD-L1 expression contributes to patients with early-stage cancer achieving a better clinical prognosis; in contrast, Yagi et al. 11 demonstrated that high PD-L1 expression was among high-risk factors for patients with EC, and preoperative treatment tended to upregulate PD-L1 expression.The bias may be due to differences in subjects, pre and postoperative treatments, antibodies, and evaluation methods. 10,11In this regard, we excluded the tremendous impact of neoadjuvant chemotherapy and adjuvant therapy on TME and patient clinical outcomes and objectively evaluated PD-L1 TC using standardized evaluation methods (staining intensity and proportion).The positive expression rate of intratumoral PD-L1 in TC was 27.8%, which was closely associated with tumor depth, stage, and clinical outcomes.Compared with the negative PD-L1 TC group, the positive group showed a 19.8% decrease in long-term survival, suggesting that PD-L1 TCs are associated with poor survival and participate in the development and progression of ESCC.
PD-L1, apart from its expression in malignant TC, has been detected in tumor-infiltrating immune cells.However, their clinical significance is not identical. 25,27e evaluated intratumoral PD-L1 TC and PD-L1 TIIC in ESCC and found the association of only PD-L1 TC with unfavorable clinical outcomes.Although Johnson et al. 26 reported the dual roles of PD-L1 in immune cells, the mechanism of PD-L1 TIIC action in ESCC remains unknown.The crucial role of bidirectional PD-L1 signaling in tumor immunity merits further investigation.TILs, which mediate key antitumor immune responses, constitute a specific cell population in the TME.Intratumoral CD8 + T cells act as cytotoxic killer cells and eliminate TC.In the present study, the 5-year survival rate of the CD8 positive group was higher than that of the negative group, along with serving as an independent favorable prognostic factor for ESCC.Unlike tumor eliminating CD8 + T cells, the clinical significance of CD4 + T cells is controversial. 33,34Mumberg and Bos et al. reported that cytokines, such as IL-2, IFN-γ, and TNF-α produced by activated CD4 + T cells can initiate and activate CD8 + T cells, and promote CD8 + T cells proliferation to inhibit tumor growth. 35,36However, the ability of CD4 + TILs to directly alter the tumor-host biological was not found in our study.This discrepancy may be due to the heterogeneity of the CD4 + T cells phenotype.Immunosuppressive Tregs differentiated from CD4 + T cells cannot be ignored.The aggregation of tumor-infiltrating Tregs regulates immune homeostasis.Our findings revealed that the recruitment of CD4 + CD25 + FOXP3 + Tregs at the tumor site was associated with an advanced-stage and lymph node metastasis, and up to 70.9% of patients with high CD4 + CD25 + FOXP3 + Tregs infiltration showed lymph node metastasis.CD4 + CD25 + FOXP3 + Tregs are associated with poor prognosis in patients with ESCC, suggesting that CD4 + CD25 + FOXP3 + Tregs are crucial for enhancing the invasive ability of TC and facilitating tumor escape.Furthermore, the status of immune homeostasis is a promising and valuable standard for identifying the significance of disease progression. 37,38Our data showed that the CD8 + TILs/CD4 + CD25 + FOXP3 + Treg ratio was associated with improved OS and inversely correlated with tumor invasion and metastasis, suggesting that immune balance affects prognosis.However, it was not as dominant as CD8 + TILs or PD-L1 TC in the multivariate analysis.
Nabeki et al. 39 indicated that FoxP3+ Tregs are stimulated by tumor-derived pro-inflammatory cytokines, and elevated levels of FoxP3+ Tregs are significantly correlated with poor prognosis in ESCC.However, FOXP3expressing cells in tumor tissues, a functionally plastic cell population in vivo, confer unregulated properties to T cells. 40,41This suggests that FOXP3 alone is insufficient as a specific marker of tumor-infiltrating Tregs.This was confirmed in our study; opposite clinical results were obtained for Tregs labeled with FOXP3 alone and for Tregs co-labeled with CD4 and CD25.
The effect of TILs or PD-L1 on the prognosis of patients with ESCC was not a singular factor; cellular synergy interactions exert a more significant impact on the prognosis.Specific subpopulations of TIL components, infiltration numbers, and PD-L1 expression in the TME are factors required for patient stratification.The 5-year OS rate of the CD8 + /PD-L1 TC − group patients was up to 84.9%, whereas it was only 24.1% in the CD8 − /PD-L1 TC + group; lymph node metastasis was significantly higher in this group compared with the other three subgroups.This is consistent with the theory that TILs/PD-L1 are involved in mediating adaptive immune resistance in tumor immunity. 42These results on the CD8/PD-L1 TC status may be more beneficial for patient risk assessment.
In summary, as neoadjuvant therapy and immunotherapy gradually replace surgery as the first-line treatment, it has become more difficult to obtain tumor samples that are unaffected by any treatment.Our results clarify the prognostic value of tumor depth, CD8 + TILs and PD-L1 TC in patients with preoperatively untreated ESCC.Elucidating the immune mechanism of CD8 + TILs/PD-L1 TC and CD8 + TILs/CD4 + CD25 + FOXP3 + Treg ratios may provide strategies for developing immunotherapy for ESCC.

F I G U R E 1
Abbreviations: PD-L1, programmed cell death ligand 1; TC, tumor cell; TIIC, tumor-infiltrating immune cell.a Statistical significance is determined using the Chi-square test or Fisher's exact test.b

F I G U R E 2
Prognostic nomogram to predict the overall survival (OS) of patients with esophageal squamous cell carcinoma.(A) A predictive nomogram for overall survival was constructed by combining proven independent prognostic factors with UICC tumor depth (T1 + 2, T3 + 4), CD8 expression (positive, negative), and PD-L1 TC (positive, negative) expression.(B) The calibration plot for the nomogram to predict 3-year survival and observed survival.(C) Time-dependent receiver operating characteristic curves for nomogram, tumor depth, and CD8 and PD-L1 TC expression to evaluate the 3-year overall survival probability.p-values were determined using the log-rank test.
Univariate and multivariate Cox regression analysis for overall survival of patients with esophageal squamous cell carcinoma (n = 126).
The bold values indicate that the p-value is statistically significant.